Search PubMed⌕ Search

Biomedical subjects

V V Petrov

Publications and source records attributed to V V Petrov.

At least 19 recordsLinked to original sources
PubMed2001

Effect of telmisartan on angiotensin II-mediated collagen gel contraction by adult rat cardiac fibroblasts.

The possible contributions of the angiotensin receptor subtypes 1 and 2 on the angiotensin II-induced collagen gel contraction by adult rat cardiac fibroblasts were studied using the specific angiotensin receptor type 1 and 2 antagonists telmisartan and P-186, respectively. Cardiac fibroblasts (from normal male adult rats) from passage 2 were cultured to confluency and added to a hydrated collagen gel, with or without angiotensin II, angiotensin II plus telmisartan, or angiotensin II plus P-186 in Dulbecco's Modified Eagle's Medium containing 5% fetal bovine serum for 1, 2, or 3 days. Control gels containing adult rat cardiac fibroblasts showed a significant amount of contraction after 3 days of incubation, causing a contraction to 67.9 +/- 7.1% of the area after 1 day. Angiotensin II (10(-7) M) stimulated (p < or = 0.05) the contraction of collagen mediated by cardiac fibroblasts after 1, 2, or 3 days. Telmisartan (10(-7) M) completely blocked the angiotensin II-induced collagen contraction by cardiac fibroblasts. P-186 (10(-7) M) had no effect on the angiotensin II-induced collagen contraction by cardiac fibroblasts. Addition of telmisartan and P-186 alone did not affect the collagen gel contraction by cardiac fibroblasts. Our data demonstrate that the effects of angiotensin II on the collagen gel contraction by adult rat cardiac fibroblasts are angiotensin II type 1 receptor mediated because they were abolished by the specific angiotensin II type 1 receptor antagonist telmisartan but not by the specific angiotensin II type 2 receptor antagonist P-186.

Angiotensin II↗
PubMed2001

T-lymphocyte and plasma angiotensin-converting enzyme activity during enalapril and losartan administration in humans.

This study evaluated the long-term effects of the angiotensin-converting enzyme inhibitor enalapril and the angiotensin II type 1 receptor antagonist losartan on the angiotensin-converting enzyme activity in T lymphocytes and plasma in patients with essential hypertension. The study was a randomized, placebo-controlled, double-blind, crossover design. Nine patients with sitting blood pressure > or = 95 mm Hg and < or = 105 mm Hg at the end of a 4-week placebo run-in period entered the double-blind phase of the study, which consisted of three 6-week periods during which patients were treated with placebo, enalapril (20 mg, once daily), or losartan (50 mg, once daily) The angiotensin-converting enzyme activity in T lymphocytes was measured as the activity of the degradation of the substrate Hippuryl-His-Leu and as the appearance of the dipeptide His-Leu, which was quantified spectrofluorometrically. Enalapril but not losartan suppressed (p < or = 0.01) the angiotensin-converting enzyme activity in plasma, whereas it stimulated (p < or = 0.05) the angiotensin-converting enzyme activity in circulating T lymphocytes. Our data document induction of angiotensin-converting enzyme in human T lymphocytes during long-term treatment with the angiotensin-converting enzyme inhibitor enalapril. Angiotensin II receptor type 1 antagonism with losartan had no effect on plasma or lymphocytic angiotensin-converting enzyme.

Administration, Oral↗
PubMed2001

Long-term treatment with enalapril or losartan does not show antiproliferative effects in peripheral blood mononuclear cells.

The objective of this study was to evaluate the long-term effects of enalapril, an angiotensin-converting enzyme inhibitor, and losartan, an angiotensin type 1 receptor antagonist, on the proliferation of peripheral blood mononuclear cells (PBMC) in patients with essential hypertension. Nine patients with a sitting diastolic blood pressure of > 95 mmHg and < 105 mmHg at the end of a 4-week placebo run-in period entered the double-blind phase of the study, which consisted of three 6-week periods during which patients were treated with placebo, enalapril (20 mg o.d.) or losartan (50 mg o.d.) The de novo synthesis of DNA, RNA and protein in PBMC was measured by [3H]-thymidine, [3H]-uridine or [3H]-leucine incorporation, respectively. Neither enalapril nor losartan affected the proliferation of PBMC measured as de novo synthesis of DNA, RNA and protein. Our data show that proliferation was not affected during angiotensin-converting enzyme inhibition with enalapril and angiotensin receptor type 1 antagonism with losartan.

Administration, Oral↗
PubMed2001

Angiotensin II-induced stimulation of collagen secretion and production in cardiac fibroblasts is mediated via angiotensin II subtype 1 receptors.

The possible contributions of the angiotensin receptor subtypes 1 (AT1) and 2 (AT2) to angiotensin II (Ang II)-induced changes in collagen secretion and production were studied using the specific angiotensin AT1- and AT2-receptor antagonists telmisartan and P-186, respectively. Cardiac fibroblasts (from normal male adult rats) from passage 2 were cultured to confluency and incubated in the presence of 10(-10) to 10(-6) M Ang II in serum-free Dulbecco's MEM medium for 24 hours. Collagen production and secretion were assayed by'H-Proline incorporation; non-collagen production and secretion were also calculated. Ang II dose-dependently increased collagen secretion and production in rat adult cardiac fibroblasts in culture. Non-collagen secretion and production were also concentration-dependently increased by Ang II. Addition of 100 nmol/l Ang II increased (p<0.01) collagen secretion and production bv 75+/-6 (SEM)% and 113+/-23%, respectively, and non-collagen secretion and production by 65+/-6% and 57+/-16%, respectively. Pretreatment of cardiac fibroblasts with telmisartan completely blocked the Ang II-induced increase in collagen secretion (p<0.001) and production(p<0.05) and in non-collagen secretion (p<0.01) and production (p<0.01). P-186 had no effect on the Ang II-induced increase in collagen secretion and production. Addition of telmisartan and P-186 did not affect collagen secretion and production in basal cardiac fibroblasts. Our data demonstrate that the effects of Ang II on collagen secretion and production in adult rat cardiac fibroblasts in culture are AT1-receptor mediated, since they were abolished by the specific AT1-receptor antagonist, telmisartan, but not by the specific AT2-receptor antagonist, P-186.

Angiotensin II↗
PubMed2000

Functional role of charged residues in the transmembrane segments of the yeast plasma membrane H+-ATPase.

As defined by hydropathy analysis, the membrane-spanning segments of the yeast plasma membrane H(+)-ATPase contain seven negatively charged amino acids (Asp and Glu) and four positively charged amino acids (Arg and His). To explore the functional role of these residues, site-directed mutants at all 11 positions and at Glu-288, located near the cytoplasmic end of M3, have been constructed and expressed in yeast secretory vesicles. Substitutions at four of the positions (Glu-129, Glu-288, Asp-833, and Arg-857) had no significant effect on ATP hydrolysis or ATP-dependent proton pumping, substitutions at five additional positions (Arg-695, His-701, Asp-730, Asp-739, and Arg-811) led to misfolding of the ATPase and blockage at an early stage of biogenesis, and substitutions of Asp-143 allowed measurable biogenesis but nearly abolished ATP hydrolysis and proton transport. Of greatest interest were mutations of Glu-703 in M5 and Glu-803 in M8, which altered the apparent coupling between hydrolysis and transport. Three Glu-703 mutants (E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled. At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis. Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases. Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group.

Adenosine Triphosphate↗
PubMed2000

Biogenesis and function of the yeast plasma-membrane H(+)-ATPase.

One of the most abundant proteins in the yeast plasma membrane is the P-type H(+)-ATPase that pumps protons out of the cell, supplying the driving force for a wide array of H(+)-dependent cotransporters. The ATPase is a 100 kDa polypeptide, anchored in the lipid bilayer by 10 transmembrane alpha-helices. It is structurally and functionally related to the P-type Na(+),K(+)-, H(+),K(+)- and Ca(2+)-ATPases of animal cells and the H(+)-ATPases of plant cells, and it shares with them a characteristic reaction mechanism in which ATP is split to ADP and inorganic phosphate (P(i)) via a covalent beta-aspartyl phosphate intermediate. Cryoelectron microscopic images of the H(+)-ATPase of Neurospora crassa and the sarcoplasmic reticulum Ca(2+)-ATPase of animal cells have recently been obtained at 8 nm resolution. The membrane-embedded portion of the molecule, which presumably houses the cation translocation pathway, is seen to be connected via a narrow stalk to a large, multidomained cytoplasmic portion, known to contain the ATP-binding and phosphorylation sites. In parallel with the structural studies, efforts are being made to dissect structure/function relationships in several P-type ATPases by means of site-directed mutagenesis. This paper reviews three phenotypically distinct classes of mutant that have resulted from work on the yeast PMA1 H(+)-ATPase: (1) mutant ATPases that are poorly folded and retained in the endoplasmic reticulum; (2) mutants in which the conformational equilibrium has been shifted from the E(2) state, characterized by high affinity for vanadate, to the E(1) state, characterized by high affinity for ATP; and (3) mutants with altered coupling between ATP hydrolysis and proton pumping. Although much remains to be learned before the transport mechanism can be fully understood, these mutants serve to identify critical parts of the polypeptide that are required for protein folding, conformational change and H(+):ATP coupling.

Adenosine Triphosphate↗
PubMed2000

Induction of cardiac fibrosis by angiotensin II.

The possible contributions of the angiotensin receptor subtypes 1 (AT1) and 2 (AT2) to angiotensin II-induced changes in collagen secretion and production were studied using the specific angiotensin receptor AT1 and AT2 antagonists telmisartan and P-186. The role of the renin-angiotensin system and its interaction with transforming growth factor-beta 1 (TGF-beta 1) in collagen deposition in cardiac fibroblasts in relation to the development of myocardial fibrosis is also discussed. Cardiac fibroblasts (from normal male adult rats) from passage 2 were cultured to confluency and incubated in the presence of angiotensin II (ANG II) in a concentration range of 10(-10)-10(-6) M in serum-free Dulbecco's MEM medium for 24 h. Collagen production and secretion were assayed by [3H]-proline incorporation and noncollagen production and secretion were also analyzed. ANG II dose-dependently increased collagen secretion and production in rat adult cardiac fibroblasts in culture. Noncollagen secretion and production were also concentration-dependently increased by ANG II. Addition of 100 nmol/l ANG II increased (p < 0.01) collagen secretion and production by 75 +/- 6 (SEM) and 113 +/- 23%, respectively, and noncollagen secretion and production by 65 +/- 6 and 57 +/- 16%, respectively. Pretreatment of cardiac fibroblasts with telmisartan completely blocked the ANG II-induced increase in collagen secretion (p < 0.001) and production (p < 0.05) and in noncollagen secretion (p < 0.01) and production (p < 0.01). P-186 had no effect on the ANG II-induced increase in collagen secretion and production. Addition of telmisartan and P-186 did not affect collagen secretion and production in basal cardiac fibroblasts. TGF-beta 1 also concentration- and time-dependently increased the secretion and production of collagen in cardiac fibroblasts. Our data demonstrate that the effects of ANG II on collagen secretion and production in adult rat cardiac fibroblasts in culture are AT1-receptor mediated since they were abolished by the specific AT1-receptor antagonist telmisartan but not by the specific AT2-receptor antagonist P-186. The ability of ANG II to induce collagen synthesis in cardiac fibroblasts may be mediated by increased TGF-beta 1 production.

Angiotensin II↗
PubMed2000

Transforming growth factor-beta(1) induces angiotensin-converting enzyme synthesis in rat cardiac fibroblasts during their differentiation to myofibroblasts.

OBJECTIVES: Appearance of angiotensin-converting enzyme (ACE) in fibrotic tissue can be the result of the action either of one particular growth factor or of cross-talk between multiple factors. Transforming growth factor-beta1 (TGF-beta(1)) is an effective inducor of the differentiation of cultured fibroblasts to myofibroblasts, which are heterogeneous cells with different phenotypes. The present study investigated whether TGF-beta(1) is able to induce, in vitro, the differentiation of cultured fibroblasts to myofibroblasts with a phenotype containing ACE. DESIGN: Adult rat cardiac ventricular fibroblasts were obtained from hearts perfused with collagenase. Cells from second passage were always used. Rat cardiac ventricular fibroblasts were incubated with TGF-beta(1) (10 ng/ml) for seven days. Cell characterisation was performed using light microscopy and indirect immunostaining. Presence of vimentin, desmin, factor VIII, alpha-smooth muscle actin, and ACE was checked with both immunostaining and Western blotting. ACE activity was measured fluorometrically with hippuryl-histidyl-leucine as substrate. Synthesis of DNA was measured as (3)H-thymidine incorporation. RESULTS: Fibroblasts contained two types of activity of hip-his-leu degradation, namely a lisinopril-dependent activity (ACE activity) and a lisinopril-independent activity ('ACE-like' activity) which is performed by peptidase(s) other than ACE. The ACE activity constituted approximately 30% of the total activity. TGF-beta(1) induced an increase in both ACE activity and ACE protein (detected by immunoblotting) by 4.5 +/- 0.9- and 6.9 +/- 2.0-fold, respectively (p<0.05). This induction of ACE was accompanied by a profound modification of the fibroblasts phenotype, which consisted of a change in cell morphology, an enlargement of cell volume and an increase in cell protein content. TGF-beta(1) profoundly inhibited (3)H-thymidine incorporation and the number of cells in growing cultures. The induction of alpha-smooth muscle actin by TGF-beta(1) (6.8 +/- 2.8-fold increase, p<0.05) simultaneously with these modifications in cell morphology and proliferation indicates the appearance of myofibroblasts. These myofibroblasts did not contain desmin. CONCLUSION: TGF-beta(1) is able to induce the appearance of ACE in cultures of adult rat cardiac ventricular fibroblasts. The appearance of the enzyme is accompanied by the differentiation of fibroblasts to myofibroblasts.

Angiotensin II↗
PubMed1998

Opposite associations of circulating aldosterone and atrial natriuretic peptide with left ventricular diastolic function in essential hypertension.

It has been shown in animal experiments that angiotensin II and aldosterone have mitogenic effects on the cardiovascular system, whereas atrial natriuretic peptide has antimitogenic properties. The aim of the present study was to relate plasma renin activity, angiotensin II, aldosterone and atrial natriuretic peptide to left ventricular structure and function, assessed by use of imaging echocardiography and transmitral Doppler velocimetry in 73 patients with essential hypertension, World Health Organization stages I-II, aged 43 +/- 10 (s.d.) years. Left ventricular mass, wall thickness and internal diameter were not independently related to the biochemical variables, except for a weak and positive association of wall thickness with plasma aldosterone (P = 0.06). However, left ventricular early inflow peak velocity and deceleration were independently and inversely related to age (P < 0.001) and to plasma aldosterone (P < 0.01), and positively to plasma atrial natriuretic peptide (P < 0.05). Peak flow velocity during atrial contraction was positively related to plasma atrial natriuretic peptide both before (P < 0.001) and after (P < 0.05) controlling for significant covariates (age, sex and blood pressure). We conclude that circulating renin, angiotensin II, aldosterone and atrial natriuretic peptide are not independently related to left ventricular mass in essential hypertension. The inverse association of plasma aldosterone with indices of diastolic function is compatible with a stimulating effect of aldosterone on myocardial fibrosis, which is opposed by atrial natriuretic peptide. The apparently conflicting positive association of this peptide with atrial peak velocity is most likely due to stimulation of its secretion by atrial involvement.

Adult↗
PubMed1997

Reactive cysteines of the yeast plasma-membrane H+-ATPase (PMA1). Mapping the sites of inactivation by N-ethylmaleimide.

We have taken advantage of cysteine mutants described previously (Petrov, V. V., and Slayman, C. W. (1995) J. Biol. Chem. 270, 28535-28540) to map the sites at which N-ethylmaleimide (NEM) reacts with the plasma-membrane H+ATPase (PMA)1 of Saccharomyces cerevisiae. When membrane vesicles containing the ATPase were incubated with NEM, six of nine mutants with single cysteine substitutions showed sensitivity similar to the wild-type enzyme. By contrast, C221A and C532A were inactivated more slowly than the wild-type control, and the C221, 532A double mutant was completely resistant, indicating that Cys-221 and Cys-532 are NEM-reactive residues. In the presence of 10 mM MgADP, the wild-type ATPase was partially protected against NEM; parallel experiments with the C221A and C532A mutants showed that the protection occurred at Cys-532, located in or near the nucleotide-binding site. Unexpectedly, the inactivation of the C409A ATPase was approximately 4-fold more rapid than in the case of the wild-type enzyme. Experiments with double mutants made it clear that this resulted from an acidic shift in pKa and a consequent acceleration of the reaction rate at Cys-532. One simple interpretation is that substitution of Cys-409 leads to a local conformational change within the central hydrophilic domain. Consistent with this idea, the reaction of fluorescein 5'-isothiocyanate at Lys-474 was also stimulated approximately 3. 5-fold by the C409A mutation. Taken together, the results of this study provide new information about the reactivity of individual Cys residues within the ATPase and pave the way to tag specific sites for structural and functional studies of the enzyme.

Cell Membrane↗
PubMed1995

Site-directed mutagenesis of the yeast PMA1 H(+)-ATPase. Structural and functional role of cysteine residues.

The yeast plasma-membrane H(+)-ATPase contains nine cysteines, three in presumed transmembrane segments (Cys-148, Cys-312, and Cys-867) and the rest in hydrophilic regions thought to be exposed at the cytoplasmic surface (Cys-221, Cys-376, Cys-409, Cys-472, Cys-532, and Cys-569). To gather new functional and structural information, we have studied the yeast ATPase by cysteine mutagenesis. It proved possible to replace seven of the nine cysteines by alanine, one at a time, without any significant decrease in ATP hydrolysis or ATP-dependent proton pumping. In the remaining two cases (Cys-409 and Cys-472), there were small but reproducible effects; the results clearly indicated, however, that no single Cys is required for activity and that, if a disulfide bridge is formed in the yeast ATPase, it does not play an obligatory structural or functional role. Next, multiple mutants were constructed to ask how many Cys residues could be replaced simultaneously while leaving a fully functional enzyme. After substitution of all "membrane" Cys (Cys-148, Cys-312, and Cys-867) together with two non-conserved Cys located in hydrophilic regions (Cys-221 and Cys-569), there were no significant abnormalities in expression (87%) or activity (89% ATP hydrolysis/93% H+ pumping) of the mutant protein. Replacement of two additional cysteines (Cys-376 near the phosphorylation site and Cys-532, in or near the ATP-binding site) caused a drop in expression (to 54%), although the corrected hydrolytic and H+ pumping activities were still normal. When Cys-472 was also mutated, the corrected activity fell to 44% hydrolysis/47% pumping; finally, substitution of Cys-409 to give a "cysteine-free" ATPase led to a very poorly expressed and poorly active enzyme. Brief exposure of the "one-cysteine" and "two-cysteine" ATPases to trypsin revealed a normal pattern of degradation, but there was a slight impairment in the ability of vanadate to protect against proteolysis. Thus, although single Cys replacements are tolerated well by the yeast ATPase, multiple replacements are progressively more harmful, suggesting that they cause small but additive perturbations of protein folding.

Cysteine↗